1. Field of the Invention
The present invention relates to methods for inhibiting angiogenesis. Thus, the present invention relates to methods of inhibiting the growth of capillary endothelial cells which form new blood microvessels. Accordingly, the present invention is directed to treating disease states, e.g., tumors such as malignant and benign tumors, characterized by an abnormally high amount of angiogenesis.
2. Discussion of Background Information
Endothelial cell proliferation and differentiation into blood capillaries (i.e., angiogenesis) are essential for growth and development, wound healing, osteogenesis, etc. Endothelial cells in adult tissues are quiescent but rapid proliferation occurs for a limited period of time during menstruation, ovulation, reproduction, implantation, mammary gland changes during lactation, and wound healing, as discussed in COCKERILL et al., “Angiogenesis: Model and Modulators”, Int Rev Cytol, 159: 113-160 (1995); and FOLKMAN et al., “Angiogenesis”, J Biol Chem, 267:10931-10934 (1992), the disclosures of which are herein incorporated by reference in their entireties.
Angiogenesis involves the development of new and small blood vessels by budding and sprouting from larger, extant vessels, as disclosed in BECK et al., “Vascular Development: Cellular and Molecular Regulation”, FASEB J., 11:365-373 (1997); and BUSSOLINO et al., “Molecular Mechanisms of Blood Vessel Formation”, TIBS, 22:251-256 (1997), the disclosures of which are herein incorporated by reference in their entireties. In normal physiological states, angiogenesis is a tightly regulated and self-limited process, as disclosed in COCKREILL et al. (1995), cited above; and KLAGSBRUN et al., “Angiogenesis”, Peptide Growth Factors and their Receptors II, pp. 549-586 (1990), the disclosures of which are herein incorporated by reference in their entireties.
Abnormal or uncontrolled angiogenesis is a prominent feature in various disease states including diabetic retinopathy, arthritis, hemangiomas, psoriasis, etc. In addition, abnormal capillary growth is an important step during the transition from hyperplasia to neoplasia and metastasis, and is critical for the growth and maintenance of many types of benign and malignant tumors, as disclosed in FOLKMAN, “The Role of Angiogenesis in Tumor Growth”, Seminar in Cancer Biol., 3:65-71 (1992); FOLKMAN et al., “Induction of Angiogenesis During the Transition from Hyperplasia to Neoplasia”, Nature 339: 58-61 (1989); FRIEDLANDER et al., “Definition of Two Angiogenic Pathways by Distinct αv Integrins”, Science, 270:1500-1502 (1995); LIOTTA et al., “Cancer Metastasis and Angiogenesis: an Imbalance of Positive and Negative Regulation”, Cell, 64:327-336 (1991); SACLARIDES et al., “Tumor Angiogenesis and Rectal Carcinoma”, Dis. Colon Rectum, 37:921-926 (1994); and SHWEIKI et al., “Patterns of Expression of Vascular Endothelial Factor (VEGF) and VEGF Receptors in Mice Suggest a Role in Hormonally Regulated Angiogenesis”, J. Clin. Invest., 91:2235-2243 (1993), the disclosures of which are herein incorporated by reference in their entireties.
Tumor growth is angiogenesis dependent. In breast carcinoma, intratumoral endothelial cells proliferate 45 times faster than endothelial cells in adjacent benign stroma, and the rate of tumor progression correlates with increased intratumoral microvascular density. Neovascularization supports tumor growth by allowing “perfusion” of nutrients, oxygen, and waste products through a crowded cell population. In addition, endothelial cells also release important “paracrine” growth factors for tumor cells.
It has been observed that in human breast carcinomas the intratumoral endothelial cell proliferation index (mean 2.7%) is 45-fold greater than that of the surrounding benign breast, as disclosed in VARTANIAN et al. , “Correlation of Intratumoral Endothelial Cell Proliferation with Microvessel Density (Tumor Angiogenesis) and Tumor Cell Proliferation in Breast Carcinoma”, Am. J. Pathol., 144:1188-1194 (1994), the disclosure of which is herein incorporated by reference in its entirety. The value of 2.7% however is unexpectedly low, especially given the much higher intratumoral microvascular density compared to adjacent benign stroma. This has suggested that a significant component of the angiogenic process is due to endothelial cell migration, capillary budding, establishment of capillary loops, and/or neovascular remodeling, as disclosed FOLKMAN et al., “Angiogenic Factors”, Science, 235:442-447 (1987); FURCHT, “Critical Factors Controlling Angiogenesis: Cell Products, Cell Matrix, and Growth Factors”, Lab. Invest., 55:505-509 (1986); DENEKAMP, “Angiogenesis, Neovascular Proliferation and Vascular Pathophysiology as Targets for Cancer Therapy”, Br. J. Radiol., 66:181-196 (1993); and MAHADEVAN et al., “Metastasis and Angiogenesis”, Rev. Oncol., 3:97-103 (1990), the disclosures of which are herein incorporated by reference in their entireties.
The association between intratumoral microvascular density and the incidence of metastases (a process by which a cancer cell leaves a primary tumor and migrates through the blood or lymph system to a new tissue or organ, where a secondary tumor grows) has been reported for invasive breast carcinoma as well as for melanoma, prostrate carcinoma, testicular carcinoma, ovarian carcinoma, rectal carcinoma, bladder carcinoma, central nervous system tumors, multiple myeloma, non-small-cell lung carcinomas, and squamous carcinoma, as disclosed in WEIDNER, “Tumor Angiogenesis: Review of Current Applications in Tumor Prognostication”, Semin. Diagn. Pathol., 10:302-313 (1993); WEIDNER, “Prognostic Factors in Breast Carcinoma”, Curr. Obstet. Gynedcol., 7:4-9 (1995); and WEIDNER, “Malignant Breast Lesions that Mimic Benign Tumors”, Semin. Diagn. Pathol., 12:2-13 (1995), the disclosures of which are herein incorporated by reference in their entireties. Invasive breast carcinoma from patients with metastases has a mean microvessel count of 101 per 200×filed (s.d=49.3, range 16-220), whereas without metastases the corresponding value is 45 per 200×filed (s.d=21.1, range 15-100). Univariate analysis has revealed these differences are statistically significant (p=0.003).
To metastasize, a tumor cell must successfully negotiate a series of obstacles. For example, tumor cells must gain access to the vasculature from the primary tumor, survive the circulation, escape immune surveillance, localize in the microvasculature of the target organ, escape from the vasculature into the target organ, and induce angiogenesis, as disclosed in FIDLER et al., “The Biology of Cancer Invasion and Metastasis”, Adv. Cancer Res., 28:149-250 (1978); NICOLSON, “Cancer Metastasis”, Sci. Am., 240:66-76 (1979); WEISS, “Biophysical Aspects of the Metastatic Cascade”, Fundamental Aspects of Metastasis, pp. 51-70 (1976); BERNSTEIN et al., “Molecular Mediators of Interactions with Extracellular Matrix Components in Metastasis and Angiogenesis”, Curr. Opin. Oncol., 6:106-113 (1994); NAGY et al., “Pathogenesis of Tumor Stroma Generation: a Critical Role for Leaky Blood Vessels and Fibrin Deposition”, Biochim Biophys. Acta, 948:305-326 (1989); MOSCATELLI et al., “Angiogenic Factors Stimulate Plasminogen Activator and Collagenase Production by Capillary Endothelial Cells”, J. Cell Biol., 91:201a (1981); FOLKMAN, “Angiogenesis”, Thrombosis and Haemostasis, 24:583-596 (1987); LIOTTA et al., Breast Cancer: Cellular and Molecular Biology, pp. 223-238 (1988); LIOTTA et al., “The Significance of Hematogenous Tumor Cell Clumps in the Metastatic Process”, Cancer Res., 36:889-894 (1976); KERBEL et al., “Clonal Dominance of Primary Tumors by Metastatic Cells: Genetic Analysis and Biological Implications”, Cancer Surv., 7:597-629 (1988); FOLKMAN, “Tumor Angiogenesis”, Canc. Med. (Chapter 11) (1992); and SUGINO et al., “Stromal Invasion is Not Essential to Blood-borne Metastasis in Mouse Mammary Carcinoma”, Scientific Program Booklet of the Pathological Society of Great Britain and Ireland, 170th Meeting, Abstract # 161 (1995), the disclosures of which are herein incorporated by reference in their entireties. Tumor growth is angiogenesis dependent, as disclosed in FOLKMAN, “Tumor Angiogenesis: Therapeutic Implications”, N. Engl. J. Med., 285:1182-1186 (1971), the disclosure of which is herein incorporated by reference in its entirety. In addition, tumor cells and blood vessels compose an integrated ecosystem in which endothelial cells could be “switched” from a resting state to a rapidly growing state by a diffusible signal from tumor cells or associated inflammatory cells.
The direct evidence, supporting that tumor growth is angiogenesis dependent, is that various methods of inhibiting angiogenesis, which are not cytostatic to tumor cells in vitro, inhibit tumor growth in vivo, as disclosed in FOLKMAN, “Angiogenesis and its Inhibition”, Important Advances in Oncology, pp. 42-62 (1985); FOLKMAN, “Clinical Applications of Research on Angiogenesis”, N. Engl. J. Med., 333:1757-1763 (1995); HARRIS et al., “Gene Therapy Through Signal Transduction Pathways and Angiogenic Growth Factors as Therapeutic Targets in Breast Cancer”, Cancer, 74:1021-1025 (1994); INGBER et al., “Synthetic Analogues of Fumagillin that Inhibit Angiogenesis and Suppress Tumor Growth”, Nature, 348:555-557 (1990); GROSS et al., “Modulation of Solid Tumor Grown in vivo by bFGF”, Proc. Am. Assoc. Cancer Res., 31:79 (#469) (1990); HORI et al., “Suppression of Solid Tumor Growth by Immunoneutralizing Monoclonal Antibody Against Human Basic Fibroblast Growth Factor”, Cancer Res., 51:6180-6184 (1991); KIM et al., “Inhibition of Vascular Endothelial Growth Factor-induced Angiogenesis Suppresses Tumor Growth in vivo”, Nature, 362:841-844 (1993); MILLAUER et al., “Glioblastoma Growth Inhibited in vivo by a Dominant-negative Flk-1 Mutant”, Nature, 367:576-579 (1994); BROOKS, “Integrin αvβ3 Antagonists Promote Tumor Regression by Inducing Apoptosis of Angiogenic Blood Vessels”, Cell, 79:1157-1164 (1994); and NICOSIA et al., “Interactions Between Newly Formed Endothelial Channels and Carcinoma Cells in Plasma Clot Culture”, Clin. Exp. Metastasis, 4:91-104 (1986), the disclosures of which are herein incorporated by reference in their entireties.
Tumor neovascularization allows growth because the new vessels allow exchange of nutrients, oxygen and waste products by a crowded cell population for which simple diffusion of these substances across its outer surfaces is no longer adequate. In addition to this “perfusion effect”, endothelial cells also release important “paracrine” growth factors for tumor cells (e.g., bFGF/FGF-2, IGF-2, PDGF, and colony stimulating factors), as disclosed in NICOSIA (1986), cited above; RAK et al., “Progressive Loss of Sensitivity to Endothelium-derived Growth Inhibitors Expressed by Human Melanoma Cells during Disease Progression”, J. Cell Physiol., 159:245-255 (1994); and HAMADA et al., “Separable Growth and Migration Factors for Large-cell Lymphoma Cells Secreted by Microvascular Endothelial Cells Derived from Target Organs for Metastasis”, Br. J. Cancer, 66:349-354 (1992). Also, the invasive chemotactic behavior of endothelial cells at the tips of growing capillaries is facilitated by their secretion of collagenases, urokinases, and plasminogen activator, as disclosed in FOX et al., “High Levels of uPA and pA-1 are Associated with Highly Angiogenic Breast Carcinomas”, J. Pathol., 170:388a (1993); and MOSCATELLI et al., “Angiogenic Factors Stimulate Plasminogen Activator and Collagenase Production by Capillary Endothelial Cells”, J Cell Biol., 91:201a (1981), the disclosures of which are herein incorporated by reference in their entireties. These degradative enzymes facilitate spreading of tumor cells into and through the adjacent fibrin-gel matrix and connective tissue stroma. Indeed, elevated levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PA-1) in breast carcinoma have been shown to be independent predictors of poor prognosis. A significant association of uPA and PA-1 with intratumoral microvascular density however, has led to the conclusion that the poor prognosis in breast carcinomas associated with elevated uPA and PA-1 may be due to an interaction between endothelial and tumor cells using the uPA enzyme system, as disclosed in FOX et al. (1993), cited above, the disclosure of which is herein incorporated by reference in its entirety. Thus, the additive impact of the “perfusion and paracrine” tumor effects, plus the endothelial-cell derived invasion-associated enzymes, all contribute to a phase of rapid tumor growth and signal a “switch” to a potentially lethal angiogenesis phenotype.
The process of tumor neovascularization shares many features with normal wound healing, as disclosed in DVORAK, “Tumors: Wounds that do not Heal. Similarities between Tumor Stroma Generation and Wound Healing”, N. Engl. J. Med., 315:1650-1659 (1986), the disclosure of which is herein incorporated by reference in its entirety, and is likely mediated by similar and specific angiogenic molecules (e.g., VEGF), which are released by the tumor cells and/or host immune cells into the stroma or possibly mobilized from a bound inactive state within the tumor stroma (e.g., FGF-1), as disclosed in FOLKMAN et al. (1987); FOLKMAN (1995); and MOSCATELLI et al. (1981), all cited above, the disclosures of which are herein incorporated by reference in their entireties. In addition to tumor cells, inflammatory cells may also be important in tumor angiogenesis. Stimulated macrophage can secrete angiogenic factors, such as TGFα, angiotropin, TNFα, and bFGF/FGF-2, as disclosed in FOLKMAN (1995), cited above; POLVERINI et al., “Induction of Neovascularization in vivo and Endothelial Proliferation in vitro by Tumor Associated Macrophages”, Lab. Invest., 51:635-642 (1984); BAIRD et al., “Immunoreactive Fibroblast Growth Factor in Cells of Peritoneal Exudate Suggests its Identity with Macrophage-derived Growth Factor”, Biochem. Biophys. Res. Commun., 126:358-364 (1985); FRATER-SCHRODER et al., “Tumor Necrosis Factor Type a, a Potent Inhibitor of Endothelial Cell Growth in vitro, is Angiogenic in vivo”, Proc. Natl. Acad. Sci (USA), 84:5277-5281 (1987); LEIBOVICH et al., “Macrophage-induced Angiogenesis Mediated by Tumour Necrosis Factor-α”, Nature, 329:630-632 (1987); SCHREIBER et al., “Transforming Growth Factor-alpha: a More Potent Angiogenic Mediator than Epidermal Growth Factor”, Science, 232:1250-1253 (1986); HOCKEL et al., “Purified Monocyte-derived Angiogenic Substance (Angiotropin) Induces Controlled Angiogenesis Associated with Regulated Tissue Proliferation in Rabbit Skin”, J. Clin. Invest., 82:1075-1090 (1988); and FOLKMAN et al., “A Heparin-binding Angiogenic Protein—Basic Fibroblast Growth Factor—is Stored within Basement Membrane”, Am. J. Pathol., 130:393-400 (1988), the disclosures of which are herein incorporated by reference in their entireties.
Clearly, many tumors have associated macrophages, which may amplify tumor angiogenesis, especially when activated by high intratumoral lactate levels caused by tumor hypoxia, as disclosed in FOLKMAN (1988), cited above, the disclosure of which is herein incorporated by reference in its entirety. Also, some human tumors are infiltrated by mast cells, as disclosed in SMOLIN, “Lymphatic Drainage from Vascularized Rabbit Cornea”, Am J. Opthalmol., 72:147-151 (1971); and KESSLER et al., “Mast Cells and Tumor Angiogenesis”, Intern. J. Can., 18:703-709 (1976), the disclosures of which are herein incorporated by reference in their entireties. Mast cells are rich in heparin, a substance known to mobilize bFGF/FGF-2 from the extracellular matrix, protect it from degradation, and potentiates its angiogenic effects, as disclosed in THORNTON et al., “Human Endothelial Cells: Use of Heparin in Cloning and Long-term Serial Cultivation, Science, 222:623-625 (1983), the disclosure of which is herein incorporated by reference in its entirety. Furthermore, when tumors are implanted in mast cell deficient mice (W/Wv), angiogenesis and tumor growth are inhibited to less than 60% of that observed in mice having normal mast cells numbers, as disclosed in DETHLEFSEN et al., “Tumor Growth and Angiogenesis in Wild Type and Mast Cell Deficient Mice”, FASEB J., 4:A623 (1990), the disclosure of which is herein incorporated by reference in its entirety. Tumor angiogenesis and tumor growth however have increased when these mast cell deficient mice are injected with exogenous mast cells along with the original bolus of tumor cells. Finally, stimulated tumor-infiltrating lymphocytes may also play a role in tumor angiogenesis by secreting cytokines that activate other inflammatory cell types, and/or chemo-attractants for other immune cells.
Angiogenesis is a complex biochemical process, and it is often difficult to study the molecular mechanism in vivo due to interference by a multitude of factors. The exact molecular details of angiogenesis (normal or abnormal) are not fully understood. It is known, however, that the sequence of events of angiogenesis involve DNA synthesis and vascular remodeling. Angiogenesis occurs in stages that orchestrate a network of cooperative interactions which include: (I) the initiation phase, characterized by increased cell membrane permeability; (ii) progression, constituted by the production of proteolytic enzymes that degrade the extracellular matrix and promote endothelial cell migration, and the entry of cells into either a proliferative or an apoptotic response; (iii) differentiation into new vessels; and (iv) the stabilization and maturation of vessels by mediator molecules that recruit mesenchymal cells to vessel walls, as discussed in COCKERILL et al. (1995); KLAGSBRUN et al. (1990); and SHWEIKI et al. (1993), all cited above, the disclosures of which are herein incorporated by reference in their entireties.
The sequence of events of angiogenesis include (i) growth of endothelial cells from small venules lacking a muscle wall; (ii) secretion of collagenases and degradation of basement membranes and connective-tissue stroma; (iii) movement of endothelial cells toward the source of the angiogenic stimulus; (iv) proliferation of endothelial cells; (v) elongation of the endothelial sprout; (vi) joining of one sprout with another to form a capillary loop; (vii) formation of a cytoplasmic vacuole with subsequent complete lumen formation and blood flow; and (viii) deposition of a new basement membrane, as disclosed in BECK et al. (1997); and BUSSOLINO et al. (1997), both cited above, the disclosures of which are herein incorporated by reference in their entireties.
Although the exact molecular mechanisms of the angiogenic process (normal or abnormal) are currently not fully understood, it is known that inducers of angiogenesis can act directly on endothelial cells, or indirectly, via accessory cells (monocytes, mastocytes, T cells and so on).
Although the list of factor(s) and/or cell(s) causing tumor angiogenesis remains incomplete, the current leading candidates for this role include bFGF/FGF-2 and VEGF, as disclosed in KANDEL et al., “Neovascularization is Associated with a Switch to the Export of bFGF in the Multi-step Development of Fibrosarcoma”, Cell, 66:1095-1104 (1991); NGUYEN et al., “Elevated Levels of the Angiogenic Peptide Basic Fibroblast Growth Factor in Urine of Bladder Cancer Patients”, J. Natl. Cancer Inst., 85:241-242 (1993); and HORI et al., “Suppression of Solid Tumor Growth by Immunoneutralizing Monoclonal Antibody Against Human Basic Fibroblast Growth Factor”, Cancer Res., 51:6180-6184 (1991), the disclosures of which are herein incorporated by reference in their entireties. Other possible angiogenic factors include: FGF-1, TGFα, TGFβ, platelet-derived endothelial cell growth factor (PD-ECGF), vascular permeability factor (VPF), folliculostellate-derived growth factor (FSDGF), granulocyte colony stimulating factor, placental growth factor, interleukin-8, hepatocyte growth factor, angiotropin, angiogenin, and TNFα, as disclosed in FOLKMAN et al. (1995); FOLKMAN (1995); and MOSCATELLI (1981), all cited above, the disclosures of which are herein incorporated by reference in their entireties. The amino acid sequences of VEGF, VPF, and FSDGF are nearly identical and likely represent the same substrate. In fact, VEGF is often designated VPF/VEGF. It has been shown in a variety of solid tumor types that tumor cells express high levels of VEGF protein and mRNA. In contrast, tumor endothelial cells express VEGF protein but not VEGF mRNA. Yet, the same endothelial cells express high levels of mRNA for the VEGF receptors Flt-1 and KDR, indicating that the endothelial-cell staining likely reflects binding of VEGF protein secreted by adjacent tumor cells. Moreover, VEGF has shown to induce in endothelial cells expression of plasminogen activator, plasminogen activator inhibitor, interstitial collagenase, and procoagulant activity, as disclosed in BROWN et al., “Increased Expression of Vascular Permeability Factor (Vascular Endothelial Growth Factor) and its Receptors in Kidney and Bladder Carcinomas”, Am J. Pathol., 143:1255-1262 (1993), the disclosure of which is herein incorporated by reference in its entirety. VEGF promotes extravasation of plasma fibrinogen, leading to fibrin deposition within the tumor matrix, a process that promotes the ingrowth of macrophages, fibroblasts, and endothelial cells, as disclosed in SENGER et al., “Vascular Permeability Factor (VPF, VEGF) in Tumor Biology”, Can Met Rev., 12:303-324 (1993), the disclosure of which is herein incorporated by reference in its entirety. In addition, it has been suggested that VEGF and bFGF/FGF-2 act in a synergistic manner to cause tumor angiogenesis, as disclosed in GOTO et al., “Synergistic Effects of Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor on the Proliferation and Cord Formation of Bovine Capillary Endothelial Cells within Collagen Gels”, Lab. Invest., 69:508-517 (1993), the disclosure of which is herein incorporated by reference in its entirety.
Various low molecular weight, non-peptide angiogenic factors have also been reported. These include 1-butyryl-glycerol, prostaglandins E1 and E2 (PEG1 and PEG2), nicotinamide, adenosine, nitric oxide, hyaluronic acid degradation products, an arachidonic acid metabolites named 12(R)-hydroxyeicosatrienoic acid (12[R]-HETrE), 8Br-cAMP, estrogens (17β-estradiol), as disclosed in FOLKMAN et al. (1987), cited above; FOLKMAN (1995), cited above; LEIBOVICH et al., “Production of Angiogenic Activity by Human Monocytes Requires an L-arginine/nitric oxide-synthase-dependent Effector Mechanism”, Proc. Natl. Acad. Sci (USA), 91:4190-4194 (1994); LANIADO-SCHWARTZMAN et al., “Activation of Nuclear Factor κβ and Oncogene Expression by 12(R)-hydroxyeicosatrienoic acid, an Angiogenic Factor in Microvessel Endothelial Cells”, J. Biol. Chem., 269:24321-24327 (1994); BANERJEE, “Microenvironment of Endothelial Cell Growth and Regulation of Protein N-glycosylation”, Indian J. Biochem. Biophys., 25:8-13 (1988); and BANERJEE et al., “Biphasic Estrogen Response on Bovine Adrenal Medulla Capillary Endothelial Cell Adhesion, Proliferation and Tube Formation”, Mol. Cell Biochem., 177:97-105 (1997). When endothelial cells are stimulated by 12(r)-HETrE, the proto-oncogenes c-myc, c-jun, and c-fos are activated, as disclosed in LANIADO-SCHWARTZMAN et al. (1988), cited above, the disclosure of which is incorporated herein by reference in its entirety.
Inactivation of a suppressor gene resulting in loss of an angiogenic suppressor substance may allow tumor angiogenesis to proceed. Indeed, the switch to active angiogenesis and the rate of the angiogenic process are likely the net effect of both stimulatory and inhibitory factors. For example, it has been shown that inactivation of a suppressor gene during carcinogenesis results in increased angiogenesis that parallels increased tumorigenicity, as disclosed in BOND et al., “Replacement of Residues of 8-22 of Angiogenin with 7-21 of RNASE-A Selectively Affects Protein-synthesis Inhibition and Angiogenesis”, Biochemistry, 29:3341-3349 (1990); and BOUCK et al., “Coordinate Control of Anchorage Independence, Actin Cytoskeleton and Angiogenesis by Human Chromosome 1 in Hamster-human Hybrids”, Cancer Res., 46:5101-5105 (1986), the disclosures of which are herein incorporated by reference in their entireties. During this process there is a 10-fold decrease in the secretion of an angiogenesis inhibitor 140 kDa glycoprotein, thrombospondin, as disclosed in RASTINEJAD et al., “Regulation of the Activity of a New Inhibitor of Angiogenesis by a Cancer Suppressor Gene”, Cell, 56:345-355 (1989), the disclosure of which is herein incorporated by reference in its entirety.
Somatic hybrid cells produced by fusion of MCF-7 human breast carcinoma cells with normal immortalized human mammary epithelial cells are suppressed in their ability to form tumors in nude mice, as disclosed in ZAJCHOWSKI et al., “Suppression of Tumor-forming Ability and Related Traits in MCF-7 Human Breast Cancer Cells by Fusion with Immortal Mammary Epithelial Cells”, Proc. Natl. Acad. Sci (USA), 87:2314-2318 (1990), the disclosure of which is herein incorporated by reference in its entirety. The hybrids has among other traits of their normal parent cells, the ability to increase the expression of the angiogenesis inhibitor thrombospondin.
A “switch” to the angiogenic phenotype by fibroblasts cultured from Li-Fraumeni patients coincides with loss of the wild-type allele of the p53 tumor suppressor gene and reduced expression of thrombospondin-1. A novel angiogenesis inhibitor, “angiostatin” is released by the primary tumor mass of a Lewis lung carcinoma. When the primary tumor is present, metastatic tumor growth is suppressed by “angiostatin”; but, after primary tumor removal, the metastases neovascularize and grow. The “angiostatin” activity co-purifies with a 38 kDa plasminogen fragment, as disclosed in O'REILLY et al., “Angiostatin: A Novel Angiogenesis Inhibitor that Mediates the Suppression of Metastases by a Lewis Lung Carcinoma”, Cell, 79:315-328 (1994), the disclosure of which is herein incorporated by reference in its entirety. Similarly, endostatin a 20 kDa C-terminal fragment of collagen XVIII prevents the angiogenic switch in pre-malignant lesions, intervening in the rapid expansion of small tumors, or inducing the regression of a large end-stage cancers, as disclosed in O'REILLY et al. (1994), cited above; and BERGERS et al., “Effects of Angiogenesis Inhibitors on Multistage Carcinogenesis in Mice”, Science, 284:808-812 (1999), the disclosures of which are herein incorporated by reference in their entireties. Other negative regulators of endothelial proliferation include: platelet factor 4, tissue inhibitors of metalloproteinases, a 16 kDa fragment of prolactin, bFGF/FGF-2 soluble receptor, and TGFβ, as disclosed in FOLKMAN (1995), cited above, the disclosure of which is herein incorporated by reference in its entirety.
Many asparagine-linked glycoproteins such as selectins, VEGFs, integrins and their receptors have been claimed to be involved during angiogenesis. Several directly angiogenic and relatively specific growth factors have been isolated: Vascular endothelial growth factor A (VEGF-A), VEGF-B, VEGF-C and placental growth factor (PIGF) are among the best characterized. These glycoproteins display high amino acid similarity in the platelet-derived growth factor (PDGF) domain. Another class of angiogenic polypeptides includes molecules with a broad range of target cells activating either a complete (migration and proliferation) or incomplete (only migration) angiogenic process in vitro.
Vascular endothelial growth factor (VEGF) induces angiogenesis, as disclosed in KIM et al., “Inhibition of Vascular Endothelial Growth Factor-induced Angiogenesis Suppresses Tumor Growth in vivo”, Nature, 362:841-844 (1993), the disclosure of which is herein incorporated by reference in its entirety. Treatment of mice previously injected with human rhabdomyosarcoma, glioblastoma multiforme, or leiomyosarcoma cell lines with a monoclonal antibody specific for VEGF has decreased the density of tumor vessels, and inhibited the tumor growth. The antibody however, has no effect on the growth rate of tumor cells in vitro. Infection of tumor endothelial cells in vivo with a retrovirus construct encoding a dominant-negative, nonfunctional mutant VEGF receptor (flk-1) also markedly has suppressed the tumor growth, MILLAUER et al., “Glioblastoma Growth Inhibited in vivo by a Dominant-negative Flk-1 Mutant”, Nature, 367:576-579 (1994), the disclosure of which is herein incorporated by reference in its entirety.
Regarding integrins and their receptors, stimulation of some types of integrin receptors leads to angiogenesis. There are two distinct pathways that induce angiogenesis mediated by different types of integrins of the vitronectin receptor family, αvβ integrins: (1) one pathway is triggered by basic fibroblast growth factor (bFGF/FGF-2) and tumor necrosis factor α (TNF α), and requires interaction with integrins αvβ3; and (2) the other is via VEGF-A and is integrin αvβ5 -dependent, as discussed in FRIEDLANDER et al. (1995), cited above; and BROOKS et al., “Requirement of Vascular Integrin αvβ3 for Angiogenesis”, Science, 264:569-571 (1994), the disclosures of which are herein incorporated by reference in their entireties.
Recent reports indicate that induction of angiogenesis by tumor or cytokine promotes vascular cell entry into the cell cycle and expression of αvβ3 integrin. It has also suggested that a single intra-vascular injection of antagonists of αvβ3 integrin (i.e., either a cyclic peptide or monoclonal antibody) disrupts ongoing angiogenesis on the chicken chorioallantoic membrane (CAM). Integrin antagonists induce apoptosis (i.e., programmed cell death) of the proliferative angiogenic vascular cells leaving preexisting quiescent blood vessels unaffected, as disclosed in BROOKS et al., “Integrin αvβ3 Antagonists Promote Tumor Regression by Inducing Apoptosis of Angiogenic Blood Vessels”, Cell, 79:1157-1164 (1994), the disclosure of which is herein incorporated by reference in its entirety.
It is also becoming evident that there are different classes of endogenous inhibitors of endothelial cell growth and motility that work in concert with inducer molecules to control angiogenesis. Reducing the concentration of inhibitor or increasing that of inducer results in an angiogenic switch, as discussed in HANAHAN et al., “Patterns and Emerging Mechanisms of the Angiogenic Switch During Tumorigenesis”, Cell, 86:353-364 (1996), the disclosure of which is herein incorporated by reference in its entirety.
Processing of glycan chains to a “high-mannose” type or to a “complex” type has also been mentioned as being relevant to endothelial cell proliferation and differentiation, as disclosed in NGUYEN et al., “1-Deoxymannojirimycin Inhibits Capillary Tube Formation in vitro, Analysis of N-linked Oligosaccharides in Bovine Capillary Endothelial Cells”, J. Biol. Chem., 267:26157-26165 (1992); and PILI et al., “The α-glucosidase I Inhibitor Castanospermine Alters Endothelial Cell Glycosylation, Prevents Angiogenesis, and Inhibits Tumor Growth”, Cancer Res., 55:2920-2926 (1995), the disclosures of which are herein incorporated by reference in their entireties. It has been suggested across the cell types that N-linked glycoproteins are important determinants of a number of cellular functions including endothelial cell proliferation, as discussed in BANERJEE (1988), cited above; BANERJEE et al., “Is Asparagine-Linked Protein Glycosylation an Obligatory Requirement for Angiogenesis?”, Indian J. Biochem. Biophys., 30:389-394 (1993); NGUYEN et al. (1992), cited above; NGUYEN et al., “A Role of Sialyl Lewis-X/A Glycoconjugates in Capillary Morphogenesis”, Nature, 365:267-269 (1993); and PILI et al. (1995), cited above, the disclosures of which are herein incorporated by reference in their entireties.
NGUYEN et al. (1992), cited above, the disclosure of which is herein incorporated by reference in its entirety, discloses that 1-deoxymannonojirimycin, an N-glycan processing inhibitor, inhibits “hybrid” and “complex” type oligosaccharides to block capillary tube formation and tumor growth. The structure of 1-deoxymannonojirimycin is disclosed on page 516 of ELBEIN, “Inhibitors of the Biosynthesis and Processing of N-linked Oligosaccharide Chains”, Ann. Rev. Biochem., 56:497-534 (1987), the disclosure of which is herein incorporated by reference in its entirety.
PILI et al. (1995), cited above, the disclosure of which is herein incorporated by reference in its entirety, discloses that castanospermine, an N-glycan processing inhibitor, inhibits “hybrid” and “complex” type oligosaccharides to block capillary tube formation and tumor growth. The structure of castanospermine is disclosed on page 516 of ELBEIN (1987), cited above, the disclosure of which is herein incorporated by reference in its entirety.
Studies have claimed that tunicamycin, a specific inhibitor of N-linked protein glycosylation has profound effects on the surface morphology, ultrastructure, and functional properties of a primary culture of bovine aortic endothelial cells and endothelial cell monolayers, as discussed in TIGANIS et al., “Functional and Morphological Changes Induced by Tunicamycin in Dividing and Confluent Endothelial Cells”, Exp. Cell Res., 198:191-200 (1992), the disclosure of which is herein incorporated by reference in its entirety. TIGANIS et al. involves an examination of the effect of tunicamycin on dividing and confluent cells. TIGANIS et al. discloses that since a feature of tunicamycin toxicity in animals is impaired permeability of brain microvessels, an important question is whether tunicamycin has a direct effect on microvessels in vivo and if so whether glycoprotein components of the tight junctions (zonula occludens) are specifically altered. The study in TIGANIS et al. involves bovine aortic cells and the endothelial lining of blood vessels.
Glycoproteins need continuous expression of Glc3Man9GlcNAc2-PP-Dol as a pre-requisite for their structural modification. In the dolichol pathway during formation of Glc3Man9GlcNAc2-PP-Dol, the enzyme, Dol-P-Man synthase, is an essential intermediate in the elongation of Man5GlcNAc2-PP-Dol to Man9GlcNAc2-PP-Dol, and an allosteric activator of GlcNAc-1-phosphate transferase, as disclosed in CHAPMAN et al., “Structure of the Lipid-linked Oligosaccharides that Accumulate in Class B thy-1-negative Mutant Lymphomas”, Cell, 17:509-515 (1979); BANERJEE et al., “Amphomycin: Effect of the Lipopeptide Antibiotic on the Glycosylation and Extraction of Dolichyl Monophosphate in Calf Brain Membranes”, Biochemistry, 20:1561-1568 (1981); and KEAN, “Site of Stimulation by Mannosyl-P-dolichol of GlcNAc-lipid Formation by Microsomes of Embryonic Chick Retina”, Glycoconiugate J., 13:675-680 (1996), the disclosures of which are herein incorporated by reference in their entireties.
As discussed in more detail below, involvement of the dolichol-linked glycan chain in capillary endothelial cell proliferation has been documented in studies on environmental insult due to CO2 depletion, and tying up the available dolichylmonophosphate (Dol-P) with amphomycin, as discussed in BANERJEE (1988), cited above; BANERJEE, “Amphomycin Inhibits Mannosylphosphoryldolichol Synthesis by Forming a Complex with Dolichylmonophosphate”, J. Biol. Chem., 264:2024-2028 (1989); and BANERJEE, “A Recent Approach to the Study of Dolichyl Monophosphate Topology in the Rough Endoplasmic Reticulum”, Acta Biochimica Polonica, 41:275-280 (1994), the disclosures of which are herein incorporated by reference in their entireties.
Regarding the relationship between the dolichol-pathway and the growth and proliferation of the capillary endothelial cells, it is noted that during somatic cell division or under the influence of angiogenic stimulus, the cell duplicates essentially all its contents including the available asparagine-linked glycoproteins. To give a few examples, many inducers of angiogenesis such as VEGF, bFGF/FGF-2, PIGF, vascular cell adhesion molecule-1, soluble E-selectin, fibronectin, laminin, thrombospondin and many of their receptors, e.g., integrins, sialyl Lewis X as well as the capillary endothelial cell marker Factor VIII:C are asparagine-linked glycoproteins and carry N-glycan chains as a part of their structures. Building of Glc3Man9GlcNAc2-PP-Dol oligosaccharide chain on the dolichol backbone through a pyrophosphate bridge in the ER membrane is a prerequisite for the asparagine residues present in the consensus sequence Asn-X-Ser/Thr to be N-glycosylated. To address a cooperation between the protein N-glycosylation pathway and the endothelial cell growth and proliferation, cells have been (1) placed under environmental stress by CO2 depletion; (2) treated with amphomycin; and (3) stimulated with a β-agonist, isoproterenol, obtaining the following results:
Regarding CO2 depletion, N-glycosylation of proteins is increased nearly 3.5-fold, and the Km for Dol-P-Man synthase is decreased by ˜50% when capillary endothelial cells (an established cell line from the microvasculature of bovine adrenal medulla) were cultured in the absence of CO2, as disclosed in BANERJEE et al., “Endothelial Cells from Bovine Adrenal Medulla Develop Capillary-like Growth Patterns in Culture”, Proc. Natl. Acad. Sci. USA, 82:4702-4706 (1985); BANERJEE et al., “Microvascular Endothelial Cells from Bovine Adrenal Medulla—A Model for in vitro Angiogenesis”, Angiogenesis: Models, Modulators and Clinical Applications, pp. 7-18 (1998); and BANERJEE (1998), cited above, the disclosures of which are herein incorporated by reference in their entireties.
Using a non-transformed capillary endothelial cell line from bovine adrenal medulla, cells were cultured in air (i.e., the absence of 5% (v/v) CO2) and showed decreased cell adhesion, did not proliferate, and died within 24 hours of culturing, as disclosed in BANERJEE (1988), cited above, the disclosure of which is herein incorporated by reference in its entirety. Interestingly, supplementation of media with 10 mM Hepes-NaHCO3, pH 7.4, improved the cell attachment as disclosed in BANERJEE (1988), cited above, the disclosure of which is herein incorporated by reference in its entirety. It has also been shown that under these experimental conditions protein glycosylation was also increased by 4.3-fold, as discussed in BANERJEE (1988), cited above, the disclosure of which is herein incorporated by reference in its entirety. Analysis of Dol-P-Man synthase, a “key” glycosyltransferase in the Dol-P pathway, as discussed in KORNFELD et al., “Assembly of Asparagine-Linked Oligosaccharides”, Annu Rev Biochem, 54:631-664 (1985), the disclosure of which is herein incorporated by reference in its entirety, suggested that the Km for GDP-mannose was reduced by ˜32% in CO2-deprived cells without a significant change in the Vmax, but the ratio of [3H]-mannose to [14C]-leucine (a protein N-glycosylation index) was increased to 4.3, compared to those cultured normally, as discussed in BANERJEE (1988), cited above, the disclosure of which is herein incorporated by reference in its entirety.
As to treatment with amphomycin, studies with amphomycin indicated that inhibition of Glc3Man9GlcNAc2-PP-Dol (OSL) biosynthesis inhibited the endothelial cell proliferation, as disclosed in BANERJEE et al. (1993), cited above, the disclosure of which is herein incorporated by reference in its entirety. Amphomycin is an undecapeptide from Streptomyces canus whose N-terminus is blocked due to a fatty acid substitution, as disclosed in HEINEMANN et al., “Amphomycin, a New Antibiotic”, Antibiot. Chemother., 3:1239-1242 (1953); and BODANSZKY et al., “Structure of the Peptide Antibiotic Amphomycin”, J. Am. Chem. Soc., 95:2352-2357 (1973), the disclosures of which are herein incorporated by reference in their entireties. Amphomycin inhibits endothelial cell proliferation in a dose-dependent manner, as discussed in BANERJEE et al. (1993), cited above, the disclosure of which is herein incorporated by reference in its entirety. The binding of amphomycin to Dol-P in the presence of Ca2+ blocks OSL assembly by interfering with the synthesis of Dol-PP-GlcNAc, Dol-P-Man, and Dol-P-Glc, respectively, as disclosed in BANERJEE (1989), cited above; BANERJEE, “A Recent Approach to the Study of Dolichyl Monophosphate Topology in the Rough Endoplasmic Reticulum”, Acta Biochimica Polonica, 41:275-280 (1994); BANERJEE, “Amphomycin: A Tool to Study Protein N-glycosylation”, J. Biosci., 11:311-319 (1987); and BANERJEE et al., “Monoclonal Antibody to Amphomycin. A Tool to Study the Topography of Dolichol Monophosphate in the Membrane”, Carbohyd. Res., 236:301-313 (1992), the disclosures of which are herein incorporated by reference in their entireties. Thus, amphomycin is a lipopeptide which binds to Dol-P in a Ca2+-dependent manner and inhibits the synthesis of Dol-P-Man, Dol-P-Glc and Dol-PP-GlcNAc and consequently Glc3Man9GlcNAc2-PP-Dol, as discussed in BANERJEE, “Amphomycin Inhibits Mannosylphosphoryldolichol Synthesis by Forming a Complex with Dolichylmonophosphate”, J. Biol. Chem., 264:2024-2028 (1989); and BANERJEE et al. (1981), cited above, the disclosures of which are herein incorporated by reference in their entireties.
In view of the above, the observations of (1) lowering of Km for GDP-mannose for Dol-P-Man synthase activity in the ER membranes in cells grown in the absence of environmental CO2 but supplemented with 100 mM Hepes-HCO3 buffer, pH 7.4, as discussed in BANERJEE (1988), cited above, the disclosure of which is herein incorporated by reference in its entirety; and (2) retardation of cellular proliferation in the presence of amphomycin, as discussed in BANERJEE et al. (1993), cited above, the disclosure of which is herein incorporated by reference in its entirety, establish that Glc3Man9GlcNAc2-PP-Dol (OSL) is essential for normal growth and proliferation of capillary endothelial cells.
Regarding treatment with isoproterenol, it was proposed that stimulating eukaryotic cells with a β-agonist, isoproterenol, increased protein N-glycosylation by activating the dolichol-pathway, as disclosed in BANERJEE, “cAMP-Mediated Protein Phosphorylation of Microsomal Membranes Increases Mannosylphosphodolichol Synthase Activity”, Proc Natl Acad Sci (USA), 84:6389-6393 (1987), the disclosure of which is herein incorporated by reference in its entirety. Addition of either isoproterenol, or cholera toxin, or prostaglandin E1, or 8Br-cAMP in the media enhanced the capillary endothelial cell proliferation by reducing the cell doubling time by 20-55 hours. cAMP did not change the cell morphology but accelerated lumen formation, as disclosed in ELIAS et al., “Direct Arterial Vascularization of Estrogen-Induced Prolactin Secreting Anterior Pituitary Tumors”, Proc Natl Acad Sci (USA), 81:4549-4553 (1984); and DAS et al., “β-adrenoreceptors of Multiple Affinities in a Clonal Capillary Endothelial Cell Line and its Functional Implication”, Mol. Cell. Biochem., 140:49-54 (1994), the disclosures of which are herein incorporated by reference in their entireties. Pre-treatment of cells with either β1-antagonist, atenolol, or a β2-antagonist, ICI-118,551, reduced protein N-glycosylation substantially. Increased protein N-glycosylation was not due to an increase in the Dol-P pool but due to an activation of Dol-P-Man synthase by cAMP-dependent protein kinase (PKA) mediated protein phosphorylation event. This activation process of Dol-P-Man synthase was further confirmed by analyzing the PKA-deficient somatic cell mutants, as disclosed in BANERJEE et al., “Protein Kinase Type I Regulates GDP-mannose:dolichylphosphate-O-β-D-mannosyl Transferase in the ER”, FASEB J, 9:1361a (1995), the disclosure of which is herein incorporated by reference in its entirety.
BANERJEE et al. (1993), cited above, discloses that tunicamycin, a GlcNAc-1P transferase inhibitor, reduced glycosylation in control cells and in isoproterenol-treated cells. In particular, BANERJEE et al. (1993) discloses that increased protein N-glycosylation by isoproterenol in the presence of exogenous dolichol monophosphate and its reduction by tunicamycin (an inhibitor of GlcNAc-1P transferase) strongly supported the view that the response was mediated through the dolichol pathway and was not due to a simple change in the dolichol monophosphate pool.
In addition, the gene for Dol-P-Man synthase has now been cloned from six different species including S. cerevisiae, as disclosed in COLUSSI et al., “Human and Saccharomyces cerevisiae Dolichol Phosphate Mannose Synthases Represent Two Class of the Enzyme, but both Function in Schizosaccharomyces pombe”, Proc Natl Acad Sci (USA), 94: 7873-7878 (1997). It has been shown that Dol-P-Man synthase in S. cerevisiae is a structural gene and its mutation is lethal. The Dol-P-Man synthase gene carries a cAMP-dependent protein phosphorylation consensus sequence and its activity is regulated by cAMP-dependent protein kinase-mediated protein phosphorylation signal, as disclosed in ORLEAN et al., “Cloning and Sequencing of the Yeast Gene for Dolichol Phosphate Mannose Synthase, an Essential Proteins”, J. Biol. Chem., 263:17499-17507 (1988); MAZHARI-TABRIZI et al, “Cloning and Functional Expression of Glycosyl Transferases from Parasitic Protozoans by Heterologous Complementation in Yeast: the Dolichol Phosphate Mannose Synthase from Trypanosoma brucei”, Biochem. J., 316:853-858 (1996); ZIMMERMAN et al., “The Isolation of a Dol-P-Man Synthase from Ustilago maydis that Functions in Saceharomyces cerevisiae”, Yeast, 12:765-771 (1996); COLUSSI et al. (1997), cited above; BANERJEE et al. (1987), cited above; and BANERJEE, “Regulation of Mannosylphosphoryldolichol Synthase Activity by cAMP-dependent Protein Phosphorylation”, Highlights of Modern Biochemistry, pp. 379-388 (1989), the disclosures of which are herein incorporated by reference in their entireties. In particular, the sequence data reyealed that the Dol-P-Man synthase gene from all species contains one consensus phosphorylation sequence in an area equivalent to Ser-141 in S. cerevisiae. 
Using a purified recombinant Dol-P-Man synthase from yeast, it has been shown that in vitro phosphorylation of the Dol-P-Man synthase by the catalytic subunit of PKA activated the Dol-P-Man synthase activity by several fold, as disclosed in BANERJEE et al., “In vitro Phosphorylation of Recombinant Dol-P-Man Synthase from S. cerevisiea Enhances its Activity”, FASEB J, 12:A1363 (1998), the disclosure of which is herein incorporated by reference in its entirety. The increase was due to an increase in Vmax and not due to an increase in Km for GDP-mannose. Furthermore, autoradiography of the [32P]Dol-P-Man synthase with an anti-DPMS antibody as well as the western blot with an anti-phosphoserine antibody confirmed the phosphorylation of the Dol-P-Man synthase. In a recent study, Ser-141 was replaced with alanine in the dpml gene from S. cerevisiea by PCR site-directed mutagenesis with the result being a significant loss of DPMS activity in the protein expressed in E. coli, as disclosed in CARRASQUILLO et al., “Serine 141 is Essential for Dol-P-Man Synthase Activity in S. cerevisiea”, Glycobiology, 8:93a (1998), the disclosure of which is herein incorporated by reference in its entirety.
The induction of apoptosis by tunicamycin has been found in (1) Chinese hamster ovary (CHO) cell glycosylation mutants Lec9, as discussed in WALKER et al., “A Functional Link Between N-linked Glycosylation and Apoptosis in Chinese Hamster Ovary Cells”, Biochem. Biophys. Res. Commun., 250:264-270 (1998), the disclosure of which is herein incorporated by reference in its entirety; (2) SV40-transformed fibroblasts line 90VAVI, as discussed in CARLBERG et al., “Short Exposures to Tunicamycin Induce Apoptosis in SV-40 Transformed but not in Normal Human Fibroblasts”, Carcinogenesis, 17(12):2589-2596 (1996), the disclosure of which is herein incorporated by reference in its entirety; and (3) sympathetic neurons, as discussed in CHANG et al., “Specific Toxicity of Tunicamycin in Induction of Programmed Cell Death of Sympathetic Neurons”, Exp. Neurol., 137(2):210-211 (1996), the disclosure of which is herein incorporated by reference in its entirety. In addition, WALKER et al. (1998), cited above, also suggested that one endogenous signal for triggering apoptosis is due to specific alterations in the N-glycosylation pathway. The Lec9 cell mutants used in WALKER et al. (1998), cited above, exhibit altered N-linked glycan structure, underglycosylation of proteins, ca.40-fold less synthesis of Glc3Man9GlcNAc2-PP-Dol and ca.2-fold less synthesis of Man5GlcNAc2-PP-Dol than parental cells, as discussed in ROSENWALD et al., “Control of Carbohydrate Processing. Increased β1,6-branching in the N-linked Carbohydrates of Lec9 CHO Mutants Appears to Arise from a Defect in Oligosaccharide-dolichol Synthesis”, Mol. Cell. Biol., 9:914-924 (1989), the disclosure of which is herein incorporated by reference in its entirety. Therefore, a predisposed condition for apoptosis may exist in Lec9 mutants which upon treatment with 0.2 μg/ml of tunicamycin has been accelerated.
A synthetic analogue of fumagillin, a naturally secreted antibiotic of Aspergillus fumigatus fresenius inhibits endothelial cell proliferation in vitro and tumor-induced angiogenesis in vivo, as disclosed in INGBER et al., “Synthetic Analogues of Fuagillin that Inhibit Angiogenesis and Suppress Tumor Growth”, Nature, 348:555-557 (1990), the disclosure of which is herein incorporated by reference in its entirety, and consequently suppresses the tumor growth. Infusions of basic fibroblast growth factor (bFGF/FGF-2) after implanting a human colon carcinoma cell line in mice not only has increased the tumor size by two-fold but also has caused an increase in the density and branching of tumor blood vessels, GROSS et al., “Modulation of Solid Tumor Grown in vivo by bFGF”, Proc. Am. Assoc. Cancer Res., 31:79 (#469) (1990), the disclosure of which is herein incorporated by reference in its entirety. Cells lacking receptors for bFGF/FGF-2 are unresponsive to bFGF/FGF-2 in vitro, and also use of a specific antibody to bFGF/FGF-2 cause ˜70% inhibition of growth of a mouse tumor, as disclosed in HORI et al., “Suppression of Solid Tumor Growth by Immunoneutralizing Monoclonal Antibody Against Human Basic Fibroblast Growth Factor”, Cancer Res., 51:6180-6184 (1991), the disclosure of which is herein incorporated by reference in its entirety.
Clinically, angiogenesis can be used as a prognostic marker or a therapeutic target for breast cancer. As a prognostic marker, the quantification of angiogenesis has become a useful technique to predict survival, the likelihood of in situ cancer progressing, a tumor response to therapy, and presence of bone marrow micrometastases. Quantification is performed by the mean tumor microvessel density by immunohistochemistry from the most vascular field of the tumor. However, not all studies has shown a correlation between the variables mentioned above. The discrepancy is most probably due to differences in methodology. Due to these limitations, other methods to quantitate angiogenesis have been investigated. Different substances, like angiogenic factors, proteases, and adhesion molecules had been measured in tumors and blood samples from breast cancer patients. These substances include VEGF, FGF-1 & 2, transforming growth factor D1, placental growth factor, TSP, and pleiotrophin. Only VEGF has shown a consistent correlation with relapse.
Furthermore, the following documents discuss angiogenesis. U.S. Pat. No. 5,766,591 to BROOKS et al., the disclosure of which is herein incorporated by reference in its entirety, discloses inhibiting angiogenesis αvβ3 antagonists such as polypeptides, monoclonal antibodies, and α β3-specific mimetics which have the capacity to interfere with αvβ3 function. BROOKS et al. discloses that inhibition of αvβ3 results in induction of apoptosis in the neovasculature cells bearing αvβ3. BROOKS et al. discloses that cells enter the S and G2/M phase.
U.S. Pat. No. 5,760,028 to JADHAV et al., U.S. Pat. No. 5,760,029 to JADHAV et al., and U.S. Pat. No. 6,130,231 to WITYAK et al., the disclosures of which are herein incorporated by reference in their entireties, disclose several heterocyclic compounds which are αvβ3 antagonists which can be used to inhibit angiogenesis.
U.S. Pat. No. 6,096,730 to COLLINS et al. and U.S. Pat. No. 6,160,166 to COLLINS et al., the disclosures of which are herein incorporated by reference in their entireties, disclose phosphonic acid agents which inhibit angiogenesis by inducing programmed cell death (apoptosis) in human microvascular endothelial cells. COLLINS et al. also discloses suramin which arrests cells in S and G2/M phases.
U.S. Pat. No. 6,146,824 to BAR-SHAVIT, the disclosure of which is herein incorporated by reference in its entirety, discloses that a thrombin derived peptide inhibits angiogenesis and induces apoptosis.
U.S. Pat. No. 6,150,407 to TUSÉ et al., the disclosure of which is herein incorporated by reference in its entirety, discloses aromatic compounds which inhibit angiogenesis by mitotic arrest and apoptosis.
YUE et al., “2-Methoxyestradiol, an Endogenous Estrogen Metabolite, Induces Apoptosis in Endothelial Cells and Inhibits Angiogenesis: Possible Role for Stress Activated Protein Kinase Signaling Pathway and Fas Expression”, Molecular Pharmacology, Vol.51, pp. 951-962 (1997), the disclosure of which is herein incorporated by reference, discloses that 2-methoxyestradiol induces apoptosis in endothelial cells and inhibits angiogenesis.
GUO et al., “Thrombospondin 1 and Type I Repeat Peptides of Thrombospondin 1 Specifically Induce Apoptosis of Endothelial Cells”, Cancer Research, 57:1735-1743 (1997), the disclosure of which is herein incorporated by reference in its entirety, discloses that thrombospondin 1 and type I repeat peptides of thrombospondin 1 induce apoptosis of endothelial cells.
U.S. Pat. No. 5,382,514 to PASSANITI et al., the disclosure of which is herein incorporated by reference in its entirety, discloses that IL-1, a peptide, regulates endothelial cell growth via autocrine mechanisms which may lead to programmed cell death (apoptosis).
U.S. Pat. No. 5,994,309 to MAZAR et al., the disclosure of which is herein incorporated by reference, discloses a peptide compound that inhibits angiogenesis, with treated animals having signs of apoptosis.
U.S. Pat. No. 5,854,205 to O'REILLY et al., the disclosure of which is herein incorporated by reference in its entirety, discloses that endostatin protein inhibits angiogenesis.
U.S. Pat. No. 5,837,682 to FOLKMAN et al., U.S. Pat. No. 5,945,403 to FOLKMAN et al., U.S. Pat. No. 6,024,688 to FOLKMAN et al., the disclosures of which are herein incorporated by reference in their entireties, disclose inhibiting angiogenesis with angiostatin fragments which are peptides. Angiostatin is known to arrest cells in G1 phase.
U.S. Pat. No. 6,114,355 to D'AMATO, the disclosure of which is herein incorporated by reference, discloses that thalidomide inhibits angiogenesis. D'AMATO also discloses 2-methoxyestradiol which affects the S phase of cells.
U.S. Pat. No. 5,985,839 to DUPONT et al., the disclosure of which is herein incorporated by reference in its entirety, discloses inhibiting angiogenesis with shark cartilage extracts.
U.S. Pat. No. 5,830,880 to SEDLACEK et al., the disclosure of which is herein incorporated by reference in its entirety, discloses gene therapy for tumors which involves, e.g., DNA for a protein which inhibits angiogenesis.
U.S. Pat. No. 4,670,394 to POLLARD et al., the disclosure of which is herein incorporated by reference, discloses blood clotting Factor VIII:C.
Glycosylation is a means of diversifying a protein without recourse to the genome, and it has the potential to both respond and reflect environmental changes. The endoplasmic reticulum (ER) is one of the largest cell organelles, its membrane constituting over one-half of the total membrane in a cell. The ER lumen, the internal space, comprises over 10% of the cell volume. The vast structure has two essential functions. (1) Proteins destined for transport to other organelles, secretion, or expression on the cell surface are synthesized on the ER surface. During translation, they are translocated into the ER lumen through a pore in the ER membrane. Inside the organelle, they are folded, sometimes with the aid of chaperon proteins, and become glycosylated. A quality control mechanism ensures that only correctly folded proteins exit the ER. Incorrectly folded proteins are retained and ultimately degraded. (2) Synthesis of lipids and cholesterol takes place on the cytoplasmic side of the membrane, as disclosed in O'REILLY et al., “Angiostatin: A Novel Angiogenesis Inhibitor that Mediates the Suppression of Metastases by a Lewis Lung Carcinoma”, Cell, 79:315-328 (1994); BERGERS et al., “Effects of Angiogenesis Inhibitors on Multistage Carcinogenesis in Mice, Science, 284:808-812 (1999); and PAHL, “Signal Transduction from the Endoplasmic Reticulum to the Cell Nucleus”, Physiol. Rev., 79:683-701 (1999), the disclosures of which are herein incorporated by reference in their entireties.
Various conditions can interfere with ER function an these are collectively called ER stress. ER stress can arise from a disturbance in protein folding, leading to an accumulation of un-or mis-folded proteins in the organelle. Cells respond to the accumulation of unfolded proteins by increasing the transcription of genes encoding ER resident proteins. The information that the capacity of the ER chaperons has been exceeded originates in the ER lumen and is transmitted to the nucleus by an intracellular signaling pathway, the unfolded protein response (UPR). This signaling pathway utilizes several novel mechanisms, including translational attenuation and a regulated mRNA splicing step, as disclosed in PAHL (1999), cited above; REDDY et al., “Assembly, Sorting and Exit of Oligomeric Proteins from the Endoplasmic Reticulum”, Bio Essays, 20:546-554 (1998); and CHAPMAN et al., “Intracellular Signaling from the Endoplasmic Reticulum to the Nucleus”, Annu. Rev. Dev. Biol., 14:459-485 (1998), the disclosures of which are herein incorporated by reference in their entirety.
Activation of mammalian UPR is characterized in part by increased transcription of at least seven genes encoding ER molecular chaperons. These are Bip/GRP78, as disclosed in LEE, “Mammalian Stress Response: Induction of the Glucose-regulated Protein Family”, Curr. Opin. Cell Biol., 4:267-273 (1992), the disclosure of which is herein incorporated by reference in its entirety, as well as induction of C/EBP homologous protein (CHOP), a transcription factor also known as growth arrest and DNA damage gene product-153 or GADD153, as disclosed in WANG et al., “Signals from the Stressed Endoplasmic Reticulum Induce C/EBP-homologous Protein (CHOP/GADD153)”, Mol. Cell. Biol., 16:4273-4280 (1996); and WANG et al., “Cloning of Mammalian Ire1 Reveals Diversity in the ER Stress Responses”, EMBO J., 17:5708-5717 (1998), the disclosures of which are herein incorporated by reference in their entireties. Three ER transmembrane signaling proteins that are thought to be the proximal effectors of the UPR are Ern1 and 2, PERK, as disclosed in WANG et al., “Cloning of Mammalian Ire1 Reveals Diversity in the ER Stress Responses”, EMBO J., 17:5708-5717 (1988); TIRASOPHON et al., “A Stress Response Pathway from the Endoplasmic Reticulum to the Nucleus Requires a Novel Bifunctional Protein Kinase/Endoribonuclease (Ire1p) in Mammalian Cells”, Genes Dev., 12:1812-1824 (1998); and HARDING et al., “Protein Translation and Folding are Coupled by an Endoplasmic-reticulum-resident Kinase”, Nature, 397:271-274 (1999), the disclosures of which are herein incorporated by reference in their entireties.
In principle, the mechanism underlying UPR-induced ER-stress condition could indirectly impede cell-cycle progression by interfering with the proper maturation of growth factor receptors or other modulators of mitogenic signaling, as disclosed in CAI et al., “Down-Regulation of
Epidermal Growth Factor Receptor-Signaling Pathway by Binding of GRP78/BiP to the Receptor Under Glucose-Starved Stress Conditions”, Journal of Cellular Physiology, 177:282-288 (1998), the disclosure of which is herein incorporated by reference in its entirety. Alternatively, ER stress may directly induce checkpoint response that prevents cells from completing their cell division cycle under conditions that compromise the proper folding and assembly of proteins response, as disclosed in BREWER et al., “Mammalian Unfolded Protein Response Inhibits Cyclin D1 Translation and Cell-cycle Progression”, Proc. Natl. Acad. Sci (USA), 96:8505-8610 (1999); and NAKAGAWA et al., “Caspase-12 Mediates Endoplasmic-reticulum-Specific Apoptosis and Cytotoxicity by Amyloid-β”, Nature, 403:98-103 (2000), the disclosures of which are herein incorporated by reference in their entireties. Since the late 1970s there has been a clear link between sugar metabolism and the UPR, as disclosed in POUYSSEGUR et al., “Induction of Two Transformation-sensitive Membrane Polypeptides in Normal Fibroblasts by a Block in Glycoprotein Synthesis or Glucose Deprivation”, Cell, 11:941-947 (1977); SHIU et al., “Glucose Depletion Accounts for the Induction of Two Transformation-sensitive Membrane Proteins in Rous Sarcoma Virus-transformed Chick Embryo Fibroblasts”, Proc. Natl. Acad. Sci. (USA) 74:3840-3844 (1977); and PELUSO et al., “Infection with Paramyxoviruses Stimulates Synthesis of Cellular Polypeptides that are also Stimulated in Cells Transformed by Rous Sarcoma Virus or Deprived of Glucose”, Proc. Natl. Acad. Sci. (USA), 75:6120-6124 (1978); GETHING et al., “Protein Folding in the Cell”, Nature, 355:33-45 (1992); PAHL et al., “A Novel Signal Transduction Pathway from the Endoplasmic Reticulum to the Nucleus is Mediated by Transcription Factor NF-kappa B”, EMBO J., 14:2580-2588 (1995); and WATOWICH et al., “Complex Regulation of Heat Shock- and Glucose-responsive Genes in Human Cells”, Mol Cell Biol., 8:393-405 (1988), the disclosures of which are herein incorporated by reference in their entireties.
The parallel advances in the mechanisms of angiogenesis have produced a plethora of target molecules and corresponding potential drugs. There are two recognized approaches to target the angiogenesis process in the clinic: vascular targeting and anti-angiogenesis. These approaches are complementary. The first, attacks the endothelial cells directly, inducing either necrosis or apoptosis. The effect is immediate. The latter affects the growth of new vessels. The different mechanisms of vascular targeting and anti-angiogenesis suggest that they may be used synergistically in the future. For small micrometastases without established vasculature, inhibition of new vessels formation will be a major target. On the other hand, larger metastases with established vessels would be effectively managed by vascular destruction initially, followed by preventing regrowth of vessels by anti-angiogenic factors. Anti-angiogenic agents have shown to demonstrate synergy with radiotherapy and many conventional anti-cancer drugs, including tamoxifen.
Irrespective of the significant progress made in recent years in the understanding of the development of the vasculature, and discovering and/or better characterizing many factors critical to vascular development and regulation, very little is known about the molecular events that trigger the withdrawal of the endothelial cell from the cell-cycle, that subsequently regulate their differentiation to form new vessels, or that finally switch off the process. It is, however, increasingly becoming clear that it is not the number of vessels, but their biological properties that determine the progress of a solid tumor.